Macrophages (MO) are the first cells infected with HIV-1 in vivo and act as a reservoir for the virus. Cytokines are potent modulators of HIV capable of enhancing and inhibiting virus replication. Since NK cells are the first line of defense against virus-infected cells and produce a number of cytokines which are biologically active on MO, we investigated the potential of NK cells to produce cytokines capable of preventing replication of HIV in MO. We found that purified NK cells and NK cell lines treated with IL-2 are rich sources of chemokines, including those which inhibit binding to chemokine co-receptors (CCR3,CCR5)for HIV. We also found that NK cells produce high levels of IL-10 as well as IL-13, a cytokine with potent monocyte differentiating properties and HIV inhibitory activity, but do not produce IL-4. Finally, we found that NK cells produce a novel factor which prevents HIV-1 replication following virus entry in MO, but not T cells. Partial purification indicates that the inhibitory factor(s) is a small molecule of approximately 10 kD with a pI between 8.0 and 10.0. The small size and basic pI are characteristic of a chemokine, but the ability of this factor to inhibit replication of HIV, and not merely virus entry, indicate that the factor is not a chemokine blocking via binding to CCR-5. Consistent with this are our findings that antibodies to the beta chemokines, including MIP-1a, MIP-1b and RANTES, are unable to reverse the HIV-1-inhibitory effect when the NK cell-derived factor is added to MO post infection. In contrast, these antibodies reverse the inhibitory effects that occur when this factor is added during virus adsorption. Taken together, our data suggest that the novel NK cell factor is distinct from beta chemokines known to inhibit HIV entry and that NK cells may play a greater role than anticipated in the regulation of HIV-1 expression in human MO. Interferon-alpha (IFN-a) has potent anti-retroviral activity in acute and chronic HIV-1 infection systems in vitro and its clinical utility has been shown in AIDS patients having high numbers of CD4+ T cells. However, IFN-a species vary in their ability to inhibit HIV replication in vitro, which may correlate with varying effects in vivo. Current studies will determine whether: 1) IFN-a components or recombinant hybrid species vary in their ability to inhibit acute HIV-1 infections of MO and T cells; 2) anti-HIV activity is associated with expression of inducible nitric oxide synthase; 3) anti-HIV activity correlates with anti-proliferative activity; and 4) whether a species of IFN-a with low anti-proliferative activity and high anti-HIV activity can be identified, which may cause fewer toxic side effects when used clinically.